Biarsenical fluorescent probes for multifunctional site-specific modification of proteins applicable in life sciences: An overview and future outlook
The cornerstone of modern molecular biology is protein fluorescent modification. In a perfect setting, it should be easy to perform, inexpensive, efficient and site-selective. Modification of proteins of interest (POI) in living cells is desired to study their behaviour and functions in the natural environment. Although multiple chemical and biological methods have been developed, only a few of them are applicable for cellular studies due to possessing appropriate physical, chemical and biological characteristics. The lack of commercialized products is definitely another problem. Historically the most applicable method for selective protein modification in live cells is based on the placement of fluorescent protein fusions to target the protein at its termini. Although this method has many advantages, such as genetic encoding, it has some drawbacks deriving mostly from the large size of the reporter and problems in the stability and maturation or folding of fused proteins. Alternative methods are based on adding to the POI self-labelling and enzyme processing tags. From all of them, only SNAP and Halo tags are successfully used in living cells. However, due to their large size, they demonstrate similar disadvantages to FPs. The last group of labels added to POI comprises short affinity tags. To date, only the tetracysteine tag/motif and its selective biarsenical binders (e.g. FlAsH and ReAsH) are widely applied in all life sciences. Since its discovery in 1998 by Tsien and co-workers this method has been enhanced and revolutionized in terms of its efficiency, formed complex stability and breadth of application. Here, we overview the whole field of knowledge, while placing most emphasis on the recent reports. We showcase the improvements of classical biarsenical probes with various optical properties as well as multifunctional molecules that add new characteristics to proteins. We also present the evolution of affinity tags and motifs of biarsenical probes demonstrating much more possibilities in use compared to FPs and other methods used already for cellular applications. We aimed to present various protocols and user observations so both beginners and advanced users of biarsenical dyes can benefit from the collected knowledge to troubleshoot their experiments. Finally, we added an outlook that hopefully will encourage more researchers to use these magnificent dyes.